Interpretive Summary: Meiotic competence of oocytes dependent on the level of follicular/oocyte maturation, is likely expressed though factors produced by oocytes and somatic cells in the follicular environment. Because of our lack of knowledge of the factors controlling porcine oocyte maturation, effective methods control meiotic competence and induce superovulation to increase numbers of quality embryos have been elusive in swine. The purposes of thi study were to 1) investigate the extent of apoptotic cell death during culture of one follicular somatic cell type, granulosa cells; 2) determine if hormones or growth factors could protect cells from this form of cell death; and 3) compare two methods of measuring apoptosis, DNA fluorescence flow cytometry and analysis of internucleosomal DNA fragments. Both methods of measuring apoptosis were in good agreement showing that granulosa cells underwent apoptosis in culture and that the presence of follicle- stimulating hormone (FSH) or insulin-like growth factor-I (IGF-I) reduced the amount of apoptosis by 50%. The results of this research will be useful to other scientists showing that the addition of FSH and IGF-I can maintain granulosa cell function in culture for the potential benefit of the oocyte.

Technical Abstract:
In experiment 1, thymocytes and granulosa cells (GC) of 3-6 mm follicles were analyzed to determine the extent of apoptosis in culture and the effect of dexamethasone (DEX). Thymocytes and GC were cultured for 3 to 48 h in medium containing 10% fetal bovine serum (FBS). Experiment 2 was conducted to determine whether porcine FSH (SUPER OV) and IGF-I attenuated apoptosis in GC and to compare two different methods of measuring apoptosis; 1) DNA fluorescence flow cytometry and 2) analysis of internucleosomal DNA fragments. GC were cultured for 24 h in medium containing 1% FBS. In experiment 1, percentage of apoptotic (%Ao) thymocytes and GC increased (p <.01) from 0.6 to 36% and from 6.1 to 68%, respectively, between 0 and 48 h. In thymocytes, 0.1 or 1.0 uM DEX caused a 33% further increase (p < .01) in apoptosis, but had no effect in GC. In experiment 2, 24 h culture resulted in an increase (p < .01) in granulosa cell apoptosis. The %Ao GC and amount of internucleosomal DNA fragments decreased (p < .01) by 50% during culture in the presence of FSH (4 NIH-S1 mU/ml) or IGF-I (50 ng/ml). The %Ao GC and amount of internucleosomal DNA fragments were positively correlated (r = 0.829, n=44, p <.0001). These results show the following: 1) apoptosis in cultured porcine GC and thymocytes followed a pattern previously established in laboratory species and could be used as model systems to investigate factors regulating apoptosis, 2) two methods of analyzing apoptosis were in excellent agreement, and 3) FSH and IGF-I attenuated spontaneous apoptosis in cultured porcine GC.